HIV and related lentiviruses have evolved essential regulatory mechanisms that utilize RNA-binding proteins to control gene expression. One protein, Tat, enhances transcription elongation by binding to the TAR RNA site at the 5' end of the viral transcripts. While much is known about its transcriptional mechanism, the structure of only a small part of the Tat-TAR complex is known. We wish to obtain a more complete structural view of lentiviral Tat-TAR complexes, to understand how such interactions can evolve in a viral context, and to utilize this knowledge to identify high affinity HIV-1 TAR binders that may inhibit Tat function and viral replication. During the previous grant period, we discovered that the arginine-rich motif (ARM) of Jembrana disease virus (JDV) Tat is a "chameleon" RNA-binding domain that can bind HIV and bovine immunodeficiency virus (BIV) TARs in two different binding modes - adopting a high affinity beta-hairpin conformation on BIV TAR and an extended conformation on HIV TAR that depends on a cellular protein, cyclin T1 (CycT1). We have developed a viral replication assay to study the evolution of Tat-TAR interactions and a bacterial assay to screen large combinatorial peptide libraries for RNA binders, both of which will aid in identifying novel high affinity HIV TAR binders. In addition, we have characterized the domain structure of CycT1, allowing us to study in more detail how the HIV TAR loop is recognized. We now plan to: 1) Utilize combinatorial libraries of RNA-binding peptides to examine the co-evolution of Tat- TAR interactions and design beta-hairpin peptides that bind HIV TAR with high affinity; 2) Test the ability of TAR binding peptides and elongation inhibitors to inhibit Tat-mediated activation and viral replication; 3) Characterize CycT1-TAR interactions and determine the structures of CycT1 or relevant complexes; 4) Generate metal-independent Tat-cyclin T1-TAR complexes to circumvent a major roadblock to biophysical and structural studies. Our proposed experiments will provide much more detailed structural insights into TAR recognition, will help us understand the structural and evolutionary relationships between the human and bovine Tat-TAR complexes and how new RNA-binding specificities can evolve, and will provide new avenues for inhibitor design.